The goals of this project involve understanding the mechanism of action of isoniazid, one of the most widely used antibiotics in the treatment of tuberculosis. Current studies are aimed at understanding the major target for isoniazid in pathogenic mycobacteria and in understanding the means by which resistance to this antibiotic is acquired in clinical isolates as well as the basis for the inherent resistance of organisms of the Mycobacterium avium complex. Recent work has focused on the role of InhA (a putative target in relatively isoniazid-insensitive saprophytic mycobacteria) in the uniquely sensitive pathogenic mycobacteria. These studies have led to the conclusion that InhA is not directly involved in the mechanism of action of isoniazid in pathogenic bacilli. We have identified a protein which is uniquely upregulated in M. tuberculosis in response to short-term, low-level exposures to isoniazid in vitro. Using biochemical techniques we have shown that this protein is an exceptionally large acyl carrier protein and is likely to be involved in mycolic acid biosynthesis. This protein is directly labeled by radioactive isoniazid and accumulates carrying lipids of a characteristic length. N-terminal amino acid sequence has identified this protein as a homolog of a smaller acyl carrier protein, FabC. We have also developed as assay for KatG activation of isoniazid based directly on metabolism of radioactive isoniazid. This assay has allowed us to screen a wide range of clinical isolates with the resulting conclusion that the major mechanism for the development of isoniazid resistance in clinical isolates is loss of KatG. The loss of KatG would be expected to severely impair such organisms with respect to growth in vivo, however, isoniazid-resistant organisms retain virulence. This paradox led us to investigate other metabolic changes occurring in such strains which led to the identification by comparative 2-dimensional gel electrophoresis of AhpC, a protein upregulated uniquely in isoniazid-resistant, KatG-impaired strains. AhpC is a homolog of a thioredoxin-dependent alkyl hydroperoxidase which functions to protect Mycobacterium tuberculosis from lethal challenge with organic hydroperoxides when overexpressed. Thus, the upregulation of AhpC appears to be a compensatory mutation which allows KatG-impaired strains to survive during an in vivo infection.